Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 March 2021 doi:10.20944/preprints202103.0108.v1 Research Article: Clinical Report Prenatal identification of a novel Mutation in the MCPH1 Gene associated with autosomal recessive primary microcephaly (MCPH) using Next Generation Sequencing (NGS). Ioannis Papoulidis1*, Makarios Eleftheriades2*, Emmanouil Manolakos 1,3, Simoni Marina Liappi1, Anastasia Konstantinidou4, Maria Papamichail5, Vassilios Papadopoulos6, Antonios Garas7, Sotirios Sotiriou8, Ioannis Papastefanou9, Georgios Daskalakis 10, Aleksandar Ristic 11 *Equal contribution / Shared co-first authorship 1. Access To Genome P.C., Clinical Laboratory Genetics, Athens-Thessaloniki, Greece. [email protected], [email protected] 2. Second Department of Obstetrics and Gynaecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece [email protected]. 3. Department of Medical Genetics, University of Cagliari, Binaghi Hospital, Cagliari, Italy. [email protected] 4. 1st Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Greece. 5. Postgraduate Programme “Maternal Fetal Medicine” Medical School National & Kapodistrian University of Athens, Greece, [email protected] 6. Department of Obstetrics & Gynecology, University of Patra, Greece. [email protected] 7. Department of Gynecology, Larissa Medical School, University of Thessaly, Larissa, Greece. [email protected] 8. Department of Clinical Embryology, Larissa Medical School, University of Thessaly, Larissa 41334, Greece. [email protected] 9. Fetal Medicine Clinic, Monis Petraki 4, Kolonaki, Athens, Greece,11521. [email protected] © 2021 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 March 2021 doi:10.20944/preprints202103.0108.v1 10. First Department of Obstetrics and Gynaecology, "Alexandra" Maternity Hospital, Medical School, National and Kapodistrian University of Athens, Athens , Greece. [email protected] 11. Obstetric and Gynecological Clinic Narodni Front, Belgrade, Serbia [email protected] Short title: Papoulidis et al. PRENATAL IDENTIFICATION OF A NOVEL MUTATION IN THE MCPH1 GENE Corresponding authors: Emmanouil Manolakos, Access to Genome – ATG Labs, 8 Sisini St., 115 28 Athens, Greece [email protected] 0030 210 7233323 Makarios Eleftheriades, Second Department of Obstetrics and Gynaecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, 76 Vas. Sofias Avenue 11 528, Athens Greece. [email protected], 0030 6944223060 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 March 2021 doi:10.20944/preprints202103.0108.v1 Established Facts and Novel Insights Established Facts • The Microcephalin protein is a pleiotropic factor playing an important role in neurogenesis; it regulates the division of neuroprogenitor cells preventing them to exhaust. • There are two categories of microcephaly: the first one is obvious during childbirth called primary microcephaly (MCPH) while the second one developes later in life and is called secondary microcephaly. Novel Insights • Since the mutation c.348del has never been associated with microcephaly phenotypes, we reveal new findings of the disease spectrum linked to MCPH1 gene mutations and its prenatal diagnosis. • Using Next Generation Sequencing (NGS) analysis, the present study reveales a new homozygous mutation of MCPH1 gene, thus expanding the disease spectrum of MCPH1 gene. Abstract MCPH1, otherwise known as the microcephalin gene (*607117) and protein, is a basic regulator of chromosome condensation (BCRT-BRCA1 C-terminus). The Microcephalin protein is made up of three BCRT domains and conserved tandem repeats of interacting phospho-peptide. There is a strong connection between mutations of the MCPH1 and reduced brain growth. Specifically, individuals with such mutations have underdeveloped brains which means smaller size, varying levels of mental retardation, delayed speech and poor language skills, individuals with mild microcephaly and normal intelligence notwithstanding. In this case, a fetus with novel homozygous mutation of the MCPH1 gene ((c.348del)), whose parents were recessive heterozygous for (c.348del), displayed severe microcephaly at 22 weeks of gestation. Due to the effect on splice sites in introns, this mutation causes forming of dysfunctional proteins which lack crucial domains of the C-terminus. Our findings portray an association between the new MCPH1 mutation ((c.348del)) and the clinical features of autosomal recessive primary microcephaly (MCPH) contributing to a broader spectrum related to these pathologies. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 March 2021 doi:10.20944/preprints202103.0108.v1 Keywords: microcephalin gene, homozygous, next generation sequencing, microcephaly. MCPH1 Abbreviations: NGS, next generation sequencing; MCPH1, Microcephalin protein Conflict of Interest: Nil Source of support: None Introduction Autosomal recessive primary microcephaly (MCPH) is an uncommon genetically heterogenous disorder of neurogenic brain development. Its characteristics are decreased head circumference during childbirth with no severe irregularities of the cerebrum and varying intellectual impairment. There are two categories of microcephaly: the first one is evident during childbirth and is called primary microcephaly (MCPH) while the second one developes later in life and is called secondary microcephaly. The main difference between the two is that while MCPH is most of the times a static irregularity of development, secondary microcephaly is a dynamic neurodegenerative disorder. Patients with primary microcephaly have not severe mental issues and also do not show syndromic, neurologic or significant dysmorphias. Whole genome (WGS) or whole exome (WES) studies identified 17 OMIM genes associated with MCPH and those are: WDR62 (*613583), CDK5RAP2 (*608201), CASC5 (*609173), ASPM (*605481), CENPJ (*609279), STIL (*181590), CEP135 (*611423), CEP152 (*613529), ZNF335 (*610827), PHC1 (*602978), CDK6 (*603368), CENPE (*117143), CENPF (*600235), PLK4 (*605031), TUBGCP6 (*610053), CEP63 (*614724), NDE1 (*609449) [1]. However, the first gene known to cause MCPH is MCPH1(*607117), making 18 genes in total associated with MCPH [2-3]. The location of MCPH1/microcephalin gene (*607117) is chromosome 8p23. Cellular responses caused by DNA damage and chromosome condensation are dependent on microcephalin, the encoded protein [4]. This protein is involved in G2/M checkpoint arrest maintaining the inhibitory phosphorylation of cyclindependent kinase 1. The ability to maintain normal brain size is lost when microcephalin is absent because it causes premature mitotic entry of neuroprogenitor cells [4-6]. In this study, we reported a case of a homozygous in MCPH1 gene (c.348del) fetus diagnosed at 22 weeks of gestation. The fetus was growth restricted showing microcephaly and partial agenesis of the corpus callosum. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 March 2021 doi:10.20944/preprints202103.0108.v1 Clinical Report A 30-year-old woman was referred to our center in her second pregnancy at 24 weeks of gestation, complicated with microcephaly (GW), just as her first one had previously. In the first pregnancy, during ultrasound examination (U/S) at 12 weeks of gestation the nuchal translucency measurement was within normal limits (NT = 1.3 mm) indicating low risk for Down syndrome (1/9052). The second trimester ultrasound examination (U/S) at 24 weeks of gestation revealed Fetal Growth Restriction (FGR), microcephaly and partial agenesis of the corpus callosum, dilatation of the third ventricle, right ureter dilatation and presence of a simple unilocular cyst at the lower lobe of the right kidney. After counselling the parents opted for fetal karyotyping and an amniocentesis was performed. Standard G-banding karyotyping revealed normal karyotype and chromosomal microarray analysis did not detect any pathogenic copy number variations. After genetic counselling the pregnancy was terminated at 26 weeks of gestation and an intact male fetus was delivered. The parents decided against autopsy and no further analysis was performed at this point. The parents were of Greek origin, healthy and nonconsanguineous. In the second pregnancy, ultrasound examination (U/S) at 12 weeks of gestation revealed high risk for Down syndrome due to increased nuchal translucency measurement (NT =3.1 mm). After counselling the parents opted for fetal karyotyping and chorionic villi sampling (CVS) was performed. Standard G-banding karyotyping revealed normal karyotype and chromosomal microarray analysis did not detect any pathogenic copy number variations. During the second trimester of pregnancy, ultrasound assessment showed severe microcephaly and FGR. Following pregnancy termination, fetal autopsy, standard G-banding karyotyping, chromosomal microarray and NGS analysis were performed. Material and Methods Molecular karyotyping High-resolution molecular karyotyping was performed with an aCGH platform of 60,000 oligonucleotides (Agilent technologies). Briefly, DNA was extracted from the chorionic villus sample with Promega Maxwell 16, and it was hybridized with the human reference DNA of the same gender (Promega Biotech). The statistical test used as a parameter to estimate the number of copies was ADM-2 (provided by the DNA analytics